Cell and Developmental Biology Commons^™

Immunotherapy Resistance In Solid Tumors: Mechanisms And Potential Solutions, Daniel Lefler, Steven Manobianco, Babar Bashir

Kimmel Cancer Center Faculty Papers

While the emergence of immunotherapies has fundamentally altered the management of solid tumors, cancers exploit many complex biological mechanisms that result in resistance to these agents. These encompass a broad range of cellular activities - from modification of traditional paradigms of immunity via antigen presentation and immunoregulation to metabolic modifications and manipulation of the tumor microenvironment. Intervening on these intricate processes may provide clinical benefit in patients with solid tumors by overcoming resistance to immunotherapies, which is why it has become an area of tremendous research interest with practice-changing implications. This review details the major ways cancers avoid both natural …

Panorama Of Human Embryo-Derived Cells In Biomedicine And Progress In Their Research And Regulation, Jianchao Gao, Yaojin Peng, Wei Wei, Shuang Lu, Chenyan Gao

Bulletin of Chinese Academy of Sciences (Chinese Version)

Since the establishment of the first human diploid cell line derived from aborted fetal tissue in the 1960s, human embryoderived cells have been widely used in biomedical field and significantly contributes to improving human health. In recent years, human pluripotent stem cells, including human embryonic stem cells (hESCs), have shown great therapeutic potential in regenerative medicine, and thus received great attention from governments and the public. However, due to various factors such as history, culture, religious beliefs, ethics and morality, research and application involving human embryo-derived cells have been controversial worldwide. This study explores the history and progress of human …

Investigating Optimal Laboratory Growth Conditions Of Gracilibacillus Halotolerans In Media Supplemented With Salt, Isaac Young

Annual Research Symposium

As interest continues to grow in the field of persister cells and their morphology, there arises an ever-evolving desire to further understand specific strains of bacteria that exemplify the qualities of seemingly anomalous survival regardless of anti-bacterial treatment. In the case of the Gracilibacillus halotolerans, a halotolerant extremophile extracted from the Great Salt Lake with known persistent characteristics, uncovering its optimal growth conditions was essential for future investigations. Identifying the optimal salinity for the growth of G. halotolerans will allow us to standardize our growth methods, uncover several mechanisms of saline tolerance, and add to future investigations of persistence with …

The Transmission Of Oropharyngeal Squamous Cell Carcinoma, Kunjal Patel, Aleesha Thomas

Mako: NSU Undergraduate Student Journal

The existence of Oropharyngeal Squamous Cell Carcinoma (OPSCC) has recently been found to have correlations with the Human Papillomavirus. HPV-associated OPSCC exhibits a unique method of infection and transmission and has made this branch an emerging disease in the recent decade. This systematic review of the literature was conducted to further explore research into Oropharyngeal Squamous Cell Cancer (OPSCC). Commonly referred to as “throat cancer”, this growth originates in the oropharynx. Symptoms of this condition include sore throat, lumps in the neck, and difficulty with swallowing. OPSCC has many variants but has shown a strong association with Human Papillomavirus (HPV), …

Ksp1 Is An Autophagic Receptor Protein For The Snx4-Assisted Autophagy Of Ssn2/Med13, Sara E Hanley, Stephen D Willis, Steven J Doyle, Randy Strich, Katrina F Cooper

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Ksp1 is a casein II-like kinase whose activity prevents aberrant macroautophagy/autophagy induction in nutrient-rich conditions in yeast. Here, we describe a kinase-independent role of Ksp1 as a novel autophagic receptor protein for Ssn2/Med13, a known cargo of Snx4-assisted autophagy of transcription factors. In this pathway, a subset of conserved transcriptional regulators, Ssn2/Med13, Rim15, and Msn2, are selectively targeted for vacuolar proteolysis following nitrogen starvation, assisted by the sorting nexin heterodimer Snx4-Atg20. Here we show that phagophores also engulf Ksp1 alongside its cargo for vacuolar proteolysis. Ksp1 directly associates with Atg8 following nitrogen starvation at the interface of an Atg8-family interacting …

Ras/Mapk Signaling Mediates Adipose Tissue Control Of Ovarian Germline Survival And Ovulation In Drosophila Melanogaster, Tancia Bradshaw, Chad Simmons, Rachael Ott, Alissa Richmond Armstrong

Faculty Publications

From insects to humans, oogenesis is tightly linked to nutritional input, yet little is known about how whole organism physiology matches dietary changes with oocyte development. Considering that diet-induced adipose tissue dysfunction is associated with an increased risk for fertility problems, and other obesity-associated pathophysiologies, it is critical to decipher the cellular and molecular mechanisms linking adipose nutrient sensing to remote control of the ovary and other tissues. Our previous studies in Drosophila melanogaster have shown that amino acid sensing, via the amino acid response pathway and mTOR-mediated signaling function within adipocytes to control germline stem cell maintenance and ovulation, …

College Of Natural Sciences 2023 Year-End Publication, College Of Natural Sciences

College of Natural Sciences Newsletters and Reports

Page 1 Dean's Message
Page 3 Department Highlights
Page 4 One Day for State
Page 5 Noble Prize Winner Speaks on Campus
Page 6-7 Faculty Excellence
Page 8-9 Student Excellence
Page 10 Outreach Program
Page 10 Events and Traditions
Page 11 Connections Abroad
Page 12 Student Spotlight
Page 13 Alumni Spotlight
Page 14 First Ever Drone Day
Page 15 Grand Opening of POET Bioproducts Center
Page 16 Work Anniversaries

Mitochondrial Quality Control By Tail-Anchored Proteins, Jose M. Delgado

Dartmouth College Ph.D Dissertations

Autophagy is a lysosome-mediated pathway responsible for the degradation of unwanted cytosolic content. During autophagy, cytoplasmic components are enveloped by a newly generated vesicle (the autophagosome), trafficked to the lysosome, and degraded. The power of autophagy lies in its ability to selectively target specific substrates for degradation—a phenomenon known as selective autophagy.

Specialized proteins known as selective autophagy receptors play a crucial role in identifying and targeting autophagy cargo. Although historically overlooked as potential regulators, our understanding of autophagy receptors is evolving beyond the assumption that receptors merely bridge targets to pre-formed autophagosomal membranes. Previous insights were often based on …

Mechanistic And Engineering Studies Of Lov Photoreceptors In Yeast, Matthew M. Cleere

Dissertations, Theses, and Capstone Projects

A variety of cellular biosensors have evolved which can translate electromagnetic radiation from the environment into biological functions. Light-Oxygen-Voltage (LOV) domains in particular, are a group of photoreceptors that respond to blue wavelengths of light and can become a cellular photoswitch by converting this stimulus through their attached effector domains. For instance, the Helix-Turn-Helix (HTH) DNA-binding activity of the bacterial transcription factor EL222 and the Regulator of G-protein Signaling (RGS) in the fungal membrane associating protein BcLOV4 are both induced by blue-light sensing LOV domains. While previous work has provided the field with a structural characterization of LOV proteins in …

Targeting Strategies To Optimize The Therapeutic Potential Of Gold Compounds Against Her2-Positive Breast Cancers, Afruja Ahad

Dissertations, Theses, and Capstone Projects

The overexpression of HER2 accounts for 20-30% of breast cancer tumors and not only serves as a marker for poor predictive clinical outcomes but also as a target for treatment. Antibody-drug conjugates (ADCs) combine the selectivity of monoclonal antibodies (mAbs) with the efficacy of chemotherapeutic drugs to provide targeted treatment without toxicity to normal tissue. Most of the ADCs currently in the clinic for cancer chemotherapy are based on complex organic molecules. In contrast, the conjugation of metallodrugs to mAbs has been overlooked when there is enormous potential in this area with the resurgence of metal-based drugs as prospective cancer …

Methamphetamine-Induced Dna Double-Stranded Breaks: The Impact Of The Dopamine Transporter And Insights Into The Mechanisms Of Dna Damage In Mouse Neuro 2a Cells, Lizette Couto

Dissertations, Theses, and Capstone Projects

Methamphetamine (METH) abuse remains a global health concern, with emerging evidence highlighting its genotoxic potential. In the central nervous system METH enters dopaminergic cells primarily through the dopamine transporter (DAT), which controls the dynamics of dopamine (DA) neurotransmission by driving the reuptake of extracellular DA into the presynaptic neuronal cell. Additional effects of METH on the storage of DA in synaptic vesicles lead to the dysregulated cytosolic accumulation of DA. Previous studies have shown that after METH disrupts intracellular vesicular stores of DA, the excess DA in the cytosol is rapidly oxidized. This generates an abundance of reactive oxygen species …

Protein-Protein Interactions In Cell Cycle Proteins: An In Silico Investigation Of Two Important Players, Andriele Eichner

Dissertations, Theses, and Capstone Projects

The examination of the cell cycle carries significant implications for the biology, health, and overall existence of all living things. These implications span from the development and growth of these organisms to the aging process and cancer, as well as the potential of stem cell therapies to repair diseases and injuries. Numerous proteins of the cell cycle are essential for cellular division and proliferation and are widely conserved over the course of evolution. In this work, we aimed to investigate the molecular processes of protein-protein interactions in cell cycle proteins, centering on two key players: Cdc6 in budding yeast and …

Diversity In The Hla-I Recognition Of Hla-F Monoclonal Antibodies: Hla-F Or Hla-Ib Monospecific, Hla-E Or Hla-G Bispecific Antibodies With Or Without Hla-Ia Reactivity, Mepur Ravindranath, Narendranath Ravindranath, Carly Amato-Menker, Fatiha El Hilali, Edward J Filippone

Division of Nephrology Faculty Papers

Previous investigators have used various anti-HLA-F monoclonal antibodies (mAbs) to demonstrate that the tissue distribution of HLA-F is highly restricted. Notably, these mAbs differed in their immunodiagnostic capabilities. Specifically, mAbs Fpep1.1 and FG1 detected HLA-F intracellularly in B cells but not on the cell surface, whereas mAb 3D11 detected HLA-F on the cell surface. The presence of HLA-F on T cells was recognized by mAb FG1 but not by mAb Fpep1.1. mAb 3D11 detected HLA-F on the cell surface of activated B cells and on peripheral blood lymphocytes, but not on the normal cells. Importantly, mAb 3D11 revealed that HLA-F …

Oxidative Stress And Ion Channels In Neurodegenerative Diseases, Razan Orfali, Adnan Z. Alwatban, Rawan S. Orfali, Liz Lau, Noble Chea, Abdullah M. Alotaibi, Young-Woo Nam, Miao Zhang

Pharmacy Faculty Articles and Research

Numerous neurodegenerative diseases result from altered ion channel function and mutations. The intracellular redox status can significantly alter the gating characteristics of ion channels. Abundant neurodegenerative diseases associated with oxidative stress have been documented, including Parkinson’s, Alzheimer’s, spinocerebellar ataxia, amyotrophic lateral sclerosis, and Huntington’s disease. Reactive oxygen and nitrogen species compounds trigger posttranslational alterations that target specific sites within the subunits responsible for channel assembly. These alterations include the adjustment of cysteine residues through redox reactions induced by reactive oxygen species (ROS), nitration, and S-nitrosylation assisted by nitric oxide of tyrosine residues through peroxynitrite. Several ion channels have been directly …

Investigation Into Cardiacmyhc-Α 334–352-Specific Tcr Transgenic Mice Reveals A Role For Cytotoxic Cd4 T Cells In The Development Of Cardiac Autoimmunity, Meghna Sur, Mahima T. Rasquinha, Kiruthiga Mone, Chandirasegaran Massilamany, Ninaad Lasrado, Channabasavaiah B. Gurumurthy, Raymond A. Sobel, Jay Reddy

Jay Reddy Publications

Myocarditis is one of the major causes of heart failure in children and young adults and can lead to dilated cardiomyopathy. Lymphocytic myocarditis could result from autoreactive CD4+ and CD8+ T cells, but defining antigen specificity in disease pathogenesis is challenging. To address this issue, we generated T cell receptor (TCR) transgenic (Tg) C57BL/6J mice specific to cardiac myosin heavy chain (Myhc)-α 334–352 and found that Myhc-α-specific TCRs were expressed in both CD4⁺ and CD8⁺ T cells. To investigate if the phenotype is more pronounced in a myocarditis-susceptible genetic background, we backcrossed with A/J mice. At the fourth …

The Use Of Fecal Microbiota Transplant In Overcoming And Modulating Resistance To Anti-Pd-1 Therapy In Patients With Skin Cancer, Tahne Vongsavath, Rodd Rohmani, Kyaw Min Tun, Vignan Manne

Department of Internal Medicine Faculty Research

While immune checkpoint inhibitors have evolved into the standard of care for advanced melanoma, 40–50% of melanoma cases progress while on therapies. The relationship between bacterium and carcinogenesis is well founded, such as in H. pylori in gastric cancers, and Fusobacterium in colorectal cancers. This interplay between dysbiosis and carcinogenesis questions whether changes in the microbiome could affect treatment. Thus, FMT may find utility in modifying the efficacy of anti-PD-1. This review aims to examine the use of FMT in treatment-resistant melanoma. A literature search was performed using the keywords “fecal microbiota transplant” and “skin cancer”. Studies were reviewed for …

Multivalent Epigraph Hemagglutinin Vaccine Protects Against Influenza B Virus In Mice, Erika M. Petro-Turnquist, Brigette Corder Kampfe, Amber Gadeken, Matthew J. Pekarek, Eric A. Weaver

Nebraska Center for Virology: Faculty Publications

Influenza B virus is a respiratory pathogen that contributes to seasonal epidemics, accounts for approximately 25% of global influenza infections, and can induce severe disease in young children. While vaccination is the most commonly used method of preventing influenza infections, current vaccines only induce strain-specific responses and have suboptimal efficacy when mismatched from circulating strains. Further, two influenza B virus lineages have been described, B/Yamagatalike and B/Victoria-like, and the limited cross-reactivity between the two lineages provides an additional barrier in developing a universal influenza B virus vaccine. Here, we report a novel multivalent vaccine using computationally designed Epigraph hemagglutinin proteins …

Research Progress In High-Throughput Phenotype Testing Technology And Equipment For Engineering Cells Based On Microfluidics, Shuang Li, Xiaojie Guo, Haibo Chen, Sisi Chen, Xin Da, Zhenghui Li, Qinxiu Liu, Yi Wang, Xinhui Xing, Chong Zhang

Bulletin of Chinese Academy of Sciences (Chinese Version)

Engineering cells are a core component of green bio-manufacturing. The efficient testing and acquisition of engineering cells with the desired phenotype is a critical factor in the successful implementation of bio-manufacturing. In recent years, based on microfluidics technologies, non-culture type single-cell phenotype high-throughput test equipment and culture type phenotype high-throughput test equipment for the growth and metabolism of single cell in a bioreactor have been developed such as microdroplet and microchamber, realizing high-throughput, automated, miniaturized, and integrated testing of the phenotypes of engineering cells and providing a powerful tool for the breeding of engineering cells. This review systematically compiles the …

Research On Innovative Development Path And Policy Guarantee Of China’S Synthetic Biology Industry, Hongjun Geng, Chang Wang

Bulletin of Chinese Academy of Sciences (Chinese Version)

Synthetic biology is hailed as the third revolution in life sciences after the discovery of the DNA double helix structure and genomic technology, and is the core driving force for the leapfrog development of next-generation biomanufacturing and the future bioeconomy, opening up an opportunity window for the reshaping of the global manufacturing map. How to strategically position the synthesis biology industry and seize the strategic high ground in the industry is a core topic of interest for global science and technology powers and manufacturing powers. Based on the identification of problems in the development of China’s synthesis biology industry, this …

Recent Progress And Perspectives On Development Of Gene Editing Tools, Zixian Liu, Chengping Li, Gogo Jun-Jie Liu

Bulletin of Chinese Academy of Sciences (Chinese Version)

The gene editing field has witnessed remarkable advancements in recent years, leading to the establishment and refinement of multidimensional gene editing platforms. These innovations have enabled precise targeted gene knockout, repair, and insertion. These tools have stimulated significant progress in fundamental research, therapeutics, agriculture, environment protection, and industrial applications. In this review, we provide a comprehensive overview of the milestones in gene editing tool development and offer perspectives on potential future directions for this rapidly evolving field.

Digitalized Design Of Cell Factory Enables Green Biomanufacturing, Fanze Meng, Rui Cao, Bing Hu, Lei Qin, Chun Li

Bulletin of Chinese Academy of Sciences (Chinese Version)

Green manufacturing is a modern manufacturing mode in the consideration of both environmental impact and efficiency of resource utilization, it represents a significant component of the “new productive force”. Centered on cell factories, green biomanufacturing leverages renewable resources to efficiently produce products that conform to the concepts of low-carbon, green, and sustainable development. The design of high-performance cell factories is pivotal for achieving efficient green biomanufacturing. Nevertheless, traditional cell factory design processes are complex, time-consuming, and labor-intensive. With advancements in artificial intelligence, digital-assisted cell factory design has emerged as a solution to save time and reduce costs. By integrating technologies …

Making The Connection: How Membrane Contact Sites Have Changed Our View Of Organelle Biology, G. Voeltz, E. Sawyer, G. Hajnóczky, W. Prinz

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The view of organelles and how they operate together has changed dramatically over the last two decades. The textbook view of organelles was that they operated largely independently and were connected by vesicular trafficking and the diffusion of signals through the cytoplasm. We now know that all organelles make functional close contacts with one another, often called membrane contact sites. The study of these sites has moved to center stage in cell biology as it has become clear that they play critical roles in healthy and developing cells and during cell stress and disease states. Contact sites have important roles …

Classification Of Colorectal Cancer Using Resnet And Efficientnet Models, Abhishek Ranjan, Priyanshu Srivastva, B Prabadevi, R Sivakumar, Rahul Soangra, Shamala K. Subramaniam

Physical Therapy Faculty Articles and Research

Introduction:

Cancer is one of the most prevalent diseases from children to elderly adults. This will be deadly if not detected at an earlier stage of the cancerous cell formation, thereby increasing the mortality rate. One such cancer is colorectal cancer, caused due to abnormal growth in the rectum or colon. Early screening of colorectal cancer helps to identify these abnormal growth and can exterminate them before they turn into cancerous cells.

Aim:

Therefore, this study aims to develop a robust and efficient classification system for colorectal cancer through Convolutional Neural Networks (CNNs) on histological images.

Methods:

Despite challenges in …

Gene Editing Of Pigs To Control Influenza A Virus Infections, Taeyong Kwon, Bianca L. Artiaga, Chester D. Mcdowell, Kristin M. Whitworth, Kevin D. Wells, Randall S. Prather, Gustavo Delhon, Mark Cigan, Stephen N. White, Jamie Retallick, Natasha N. Gaudreault, Igor Morozov, Juergen A. Richt

School of Veterinary and Biomedical Sciences: Faculty Publications

Proteolytic activation of the hemagglutinin (HA) glycoprotein by host cellular proteases is pivotal for influenza A virus (IAV) infectivity. Highly pathogenic avian influenza viruses possess the multibasic cleavage site of the HA which is cleaved by ubiquitous proteases, such as furin; in contrast, the monobasic HA motif is recognized and activated by trypsin-like proteases, such as the transmembrane serine protease 2 (TMPRSS2). Here, we aimed to determine the effects of TMPRSS2 on the replication of pandemic H1N1 and H3N2 subtype IAVs in the natural host, the pig. The use of the CRISPR/Cas 9 system led to the establishment of homozygous …

A Cyclin D1 Intrinsically Disordered Domain Accesses Modified Histone Motifs To Govern Gene Transcription, Xuanmao Jiao, Gabriele Di Sante, Mathew Casimiro, Agnes Tantos, Anthony Ashton, Zhiping Li, Yen Quach, Dharmendra Bhargava, Agnese Di Rocco, Claudia Pupo, Marco Crosariol, Tamas Lazar, Peter Tompa, Chenguang Wang, Zuoren Yu, Zhao Zhang, Kawthar Aldaaysi, Ratna Vadlamudi, Monica Mann, Emmanuel Skordalakes, Andrew Kossenkov, Yanming Du, Richard Pestell

Department of Pharmacology, Physiology, and Cancer Biology Faculty Papers

The essential G₁-cyclin, CCND1, is frequently overexpressed in cancer, contributing to tumorigenesis by driving cell-cycle progression. D-type cyclins are rate-limiting regulators of G₁-S progression in mammalian cells via their ability to bind and activate CDK4 and CDK6. In addition, cyclin D1 conveys kinase-independent transcriptional functions of cyclin D1. Here we report that cyclin D1 associates with H2B^S14 via an intrinsically disordered domain (IDD). The same region of cyclin D1 was necessary for the induction of aneuploidy, induction of the DNA damage response, cyclin D1-mediated recruitment into chromatin, and CIN gene transcription. In response to …

Naturally Derived Phenethyl Isothiocyanate Modulates Induction Of Oxidative Stress Via Its N-Acetylated Cysteine Conjugated Form In Malignant Melanoma, Sotiris Kyriakou, Nikoletta Demosthenous, Tom Amery, Kyle J. Stewart, Paul G. Winyard, Rodrigo Franco, Aglaia Pappa, Mihalis I. Panayiotidis

School of Veterinary and Biomedical Sciences: Faculty Publications

Phenethyl isothiocyanate (PEITC) is a secondary metabolic product yielded upon the hydrolysis of gluconasturtiin and it is highly accumulated in the flowers of watercress. The aim of the current study was to assess the role of a naturally derived PEITC-enriched extract in the induction of oxidative stress and to evaluate its anti-melanoma potency through the regulation of its metabolism with the concurrent production of the N-acetyl cysteine conjugated by-product. For this purpose, an in vitro melanoma model was utilized consisting of human primary (A375) cells as well as metastatic (COLO-679) malignant melanoma cells together with non-tumorigenic immortalized keratinocytes (HaCaT). …

Tail-Tape-Fused Virion And Non-Virion Rna Polymerases Of A Thermophilic Virus With An Extremely Long Tail, Anastasiia Chaban, Leonid Minakhin, Ekaterina Goldobina, Brain Bae, Yue Hao, Sergei Borukhov, Leena Putzeys, Maarten Boon, Florian Kabinger, Rob Lavigne, Kira S Makarova, Eugene V Koonin, Satish K Nair, Shunsuke Tagami, Konstantin Severinov, Maria L Sokolova

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP) that defines the length of its extraordinarily long tail. Here, we show that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We report the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab-claw-like architecture. By contrast, the virion RNAP adopts a unique flat structure without a clamp. Structure and sequence comparisons of …

Microstructure-Based Modeling Of Primary Cilia Mechanics, Nima Mostafazadeh, Andrew Resnick, Y.-N. Young, Zhangli Peng

Physics Faculty Publications

A primary cilium, made of nine microtubule doublets enclosed in a cilium membrane, is a mechanosensing organelle that bends under an external mechanical load and sends an intracellular signal through transmembrane proteins activated by cilium bending. The nine microtubule doublets are the main load-bearing structural component, while the transmembrane proteins on the cilium membrane are the main sensing component. No distinction was made between these two components in all existing models, where the stress calculated from the structural component (nine microtubule doublets) was used to explain the sensing location, which may be totally misleading. For the first time, we developed …

Role Of Cyp3a5 In Prostate Cancer Growth, Ranjana Mitra, Jake Mclean, Ajay P. Singh, Edwin C. Oh

COM Scholarship

No abstract provided.

Fused In Sarcoma Regulates Glutamate Signaling And Oxidative Stress Response, Chiong-Hee Wong, Abu Rahat, Howard C Chang

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Mutations in fused in sarcoma (fust-1) are linked to ALS. However, how these ALS causative mutations alter physiological processes and lead to the onset of ALS remains largely unknown. By obtaining humanized fust-1 ALS mutations via CRISPR-CAS9, we generated a C. elegans ALS model. Homozygous fust-1 ALS mutant and fust-1 deletion animals are viable in C. elegans. This allows us to better characterize the molecular mechanisms of fust-1-dependent responses. We found FUST-1 plays a role in regulating superoxide dismutase, glutamate signaling, and oxidative stress. FUST-1 suppresses SOD-1 and VGLUT/EAT-4 in the nervous system. FUST-1 also regulates synaptic AMPA-type glutamate receptor …